Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein.
The pH-sensitive fluorescent indicator dye 2', 7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF) is routinely used to measure intracellular pH within cells. Surprisingly, no studies have been performed to see if various solution parameters modulate the fluorescence intensity of BCECF even though viscosity artifacts have been reported for particular Ca2+ selective dyes. In this report we demonstrate that even minor increases in the concentration of a number of different agents significantly decrease the excitation fluorescence intensity at two wavelengths routinely used to determine solution pH. Solution viscosity was varied using a number of different agents including glycerol, sucrose, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose. In general, there was a detectable and significant decrease in the maximum fluorescence excitation ratio as the viscosity was increased, although the effect was more dramatic with Newtonian solutions than with non-Newtonian solutions. This same general effect was seen at pH 6.5, 7.0, and 7.3, a range of pH levels where BCECF is found to be particularly sensitive. To correct for these artifactually low values we used different combinations of excitation wavelengths to determine which could be used to accurately measure pH while minimizing the artifact. Choosing excitation wavelengths so that excitation ratios were collected at 470 and 435 nm allowed a significant signal to quantitatively measure pH while the artifact was nearly abolished.